Diseases caused by pathogenic bacteria require that the microorganisms sequester iron from the host as an essential growth factor. This raises a problem for pathogens, as free iron is not readily available. Further complicating the problem for the pathogen, the host activates a number of molecules meant to interfere with access to iron stores. Most of the details of this interplay between the host and the pathogen competing for limiting iron have been devoted to understanding the biology of extracellular pathogens. In contrast, the dynamics of iron competition in an intracellular environment is poorly understood, particularly for intravacuolar pathogens. No strategies have been forwarded for how iron is transported into the pathogen replication vacuole and the source of the intracellular store of iron accessed by these pathogens is unknown. Furthermore, understanding how the host cell limits iron availability to these pathogens is limited to the analysis of one phagosomal divalent metal exporter. This application proposes to attack this problem by taking advantage of recent data on the biology of iron acquisition by Legionella pneumophila and technological advances that allow the analysis of random mutations in any cell type. The experiments described propose to identify host cell components that either facilitate or restrict L. pneumophila access to cellular stores of iron. Access to unknown stores of iron by this intravacuolar pathogen requires the function of the MavN protein, which is inserted into the membrane replication compartment surrounding the bacterium and is hypothesized to be an iron transporter. Using strains lacking this protein, mutant hunt strategies are proposed that identify host proteins that collaborate with MavN to facilitate iron access to the replication compartment or which interfere with bacterial access to this metal. The key technological advances forwarded are twofold. First, experiments will involve the use of bacterial strains harboring a transcriptional iron biosensor, to identify hst cells disrupted for pathogen iron acquisition. Secondly, the host mutants will be identified using a genome-scale lentivirus CRISPR/Cas9 mutant library, which allows lesions to be introduced permanently into any cell type sensitive to the lentivirus. A set of straightforward criteria is proposed that will allow ranking of each of the mutants for purposes of further study. Of highest priority are proteins predicted to provide the interface between organelle iron stores and the replication vacuole, as well as proteins that may play a role in restricting pathogen access to intracellular iron.